Method for improving the pharmaceutic properties of microparticles comprising diketopiperazine and an active agent

ABSTRACT

Methods are provided for drying a particle. Specifically, there is provided a spray-dried diketopiperazine-insulin particle formulation having improved aerodynamic performance and in which the active agent is more stabile and efficiently delivered as compared to that of the lyophilized diketopiperazine-insulin formulation. The dry powders have utility as pharmaceutical formulations for pulmonary delivery.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 16/453,875, filed on Jun. 26, 2019, which is acontinuation of U.S. patent application Ser. No. 16/151,707, filed Oct.4, 2018, which is a divisional of U.S. patent application Ser. No.14/971,785 filed Dec. 16, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/942,482 filed Jul. 15, 2013, which is acontinuation of U.S. patent application Ser. No. 13/239,696 filed Sep.22, 2011, which is a continuation of U.S. patent application Ser. No.11/678,046 filed Feb. 22, 2007, which claims the benefit under of U.S.Provisional Application No. 60/776,605 filed Feb. 22, 2006, the contentsof each of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to the field of dry powder pharmaceuticals. Theinvention discloses methods of obtaining particles with improvedaerodynamic performance or in which the active agent is more stable andefficiently delivered. More particularly, the present invention concernsmethods for drying, particularly spray drying diketopiperazine-insulin(DKP-insulin) particles. The dry powders of the invention have utilityas pharmaceutical formulations for pulmonary delivery.

Description of the Related Art

A number of different methodologies are employed in the art forpreparing particles as a dry powder composition. These methodologiesinclude, for example, lyophilization, evaporation, phase separation, andspray drying (see PCT Patent Application: WO 91/16038). In themanufacture of dry powder pharmaceuticals some methods start with thecomponents in solution and form the particles of the powder by removingsolvent. Other methods form particles in a separate, earlier step, suchas by precipitation, and can result in a particle in suspension, whichmust then be dried. Methods such as lyophilization and evaporation areoften used particularly for drying or removing a solvent from particlesin suspension, whereas spray drying has more typically been used forparticle formation from solution. For example, see U.S. Pat. Nos.5,976,574; 5,985,248; 6,001,336; 6,051,256; 6,077,543; 6,365,190;6,372,258; 6,423,344; 6,479,049; 6,509,006; 6,569406; 6,572,893;6,582,728; 6,838,076; and 6,896,906.

Lyophilization, or freeze drying, involves a process in which solvent,typically water, is removed from a product after it is frozen and placedunder a vacuum, allowing the ice to change directly from solid to vaporwithout passing through a liquid phase. The process consists of threeseparate, unique, and interdependent processes; freezing, primary drying(sublimation), and secondary drying (desorption). During spray drying, a(generally aqueous) solution is introduced via a nozzle (e.g., a twofluid nozzle), spinning disc, or an equivalent device into a hot gasstream. Passage through the nozzle atomizes the solution into finedroplets. The heat energy supplied by the gas stream causes theevaporation of water or other solvents, thereby producing fineparticles.

Drug delivery using substituted diketopiperazine microparticles has beendescribed in U.S. Pat. Nos. 5,352,461; 5,503,852; 6,331,318; 6,395,774and 6,663,898. Pulmonary delivery of diketopiperazine microparticles asdry powders is described in U.S. Pat. Nos. 5,503,852; 6,428,771;6,444,226 and 6,652,885. Various methods for forming and loadingdiketopiperazine particles for drug delivery are disclosed in U.S. Pat.No. 6,444,226, U.S. patent application Nos. 11/532,063 and 11/532,065both filed on Sep. 14, 2006, and U.S. Provisional patent applicationSer. No. 60/717,524, filed on Sep. 14, 2005. Each of these documents isincorporated herein by reference for all they contain regardingdiketopiperazines, diketopiperazine microparticles and their use in drugdelivery. Dry powders made according to these teachings work well forpulmonary delivery; however there remains room for improvement ofvarious pharmaceutic properties. The present invention serves toovercome the need in the art for obtaining improved particles havingsuperior aerodynamics and providing more efficient delivery and greaterstability of the active agent.

SUMMARY OF THE INVENTION

The present invention is directed to methods of obtaining an improvedparticle and/or an improved dry powder. The particles and powderscontemplated by the present invention are comprised of adiketopiperazine derivative combined with an active agent. In particularembodiments of the present invention, the particle is adiketopiperazine-insulin particle formulation having improved stability,aerodynamic properties, and pharmacodynamic properties when dried by theprocess of spray drying as compared to that of freeze drying. In otherembodiments, there is provided a spray-dried diketopiperazine-insulinparticle formulation or dry powder.

In a particular embodiment of the present invention, the particlecomprising a diketopiperazine is prepared and provided in a suspension,typically an aqueous suspension, to which a solution of the active agentis added. Active agents of the present invention may include one or moreof the following: insulin, calcitonin, parathyroid hormone 1-34, orother bioactive fragment of parathyroid hormone, octreotide, leuprolide,and RSV peptide, felbamate, cannabinoid antagonists and/or agonists,muscarinic antagonists and/or agonists, heparin, low molecular weightheparin, cromolyn, sildenafil, vardenafil, tadalafil growth hormone,zidovudine (AZT), didanosine (DDI), granulocyte-colony stimulatingfactor (GCSF), lamotrigine, chorionic gonadotropin releasing factor,luteinizing release hormone, β-galactosidase, GLP-1, exendins 1-4,ghrelin, and fragments thereof, but are not limited to such. In anotherembodiment, the active agent is a peptide or protein such as insulin oran analogue thereof.

In a particular embodiment, the active agent is insulin or an analoguethereof.

The present invention discloses methods of obtaining particles withimproved aerodynamic performance and in which the active agent is morestable and efficiently delivered. More particularly, the presentinvention concerns methods for drying, particularly spray drying,diketopiperazine-insulin particles. The dry powders have utility aspharmaceutical formulations for pulmonary delivery. In otherembodiments, the diketopiperazine-insulin dry powders may be utilizedfor nasal delivery.

Thus, in particular embodiments the present invention provides a methodof preparing a dry powder medicament with an improved pharmaceuticproperty, comprising the steps of: (a) providing a solution of adiketopiperazine; (b) providing a solution of an active agent; (c)forming particles; and (d) combining the diketopiperazine and the activeagent; and thereafter (e) removing solvent by spray drying to obtain adry powder, wherein the dry powder has an improved pharmaceutic propertyas compared to a dry powder obtained by removing solvent bylyophilization.

In another embodiment, the improved pharmaceutic property is selectedfrom the group consisting of improved stability of the active agent,increased density of the dry powder, and improved aerodynamicperformance of the dry powder. In still yet another embodiment, animproved aerodynamic performance of the dry powder is measured by thepercentage of particles in the respirable range (respirable fraction)delivered from the inhaler. The respirable fraction, as contemplated inthe present invention, may be greater than about 40% or greater thanabout 50%, or greater than about 60%, but is not limited to such.

In other embodiments of the present invention, it is contemplated thatthe insulin content of the microparticles is within the range of about3% to about 50% by weight of the dry powder formulation. In otherinstances, the insulin concentration is within the range of about 7% toabout 25% by weight. In preferred embodiments insulin content is about19.0, 19.1, 19.2 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, or 19.9% by weight.In another preferred embodiment, insulin concentration is at about 11%by weight. In still other preferred embodiments the insulin content isabout 10, 12, 13, 14, 15, 16, 17, or 18% by weight. In variousembodiments, about is defined as ±0.1, 0.2, 0.5, 1, or 2%, so long asthe uncertainty does not exceed 10% of the insulin content.

In still yet another embodiment, there is provided in the presentinvention a diketopiperazine having the formula2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl. In apreferred embodiment, the diketopiperazine is fumaryl diketopiperazine.

In yet another particular embodiment of the present invention there isprovided a dry powder prepared according to the method of preparing adry powder medicament with an improved pharmaceutic property, comprisingthe steps of: (a) providing a solution of a diketopiperazine; (b)providing a solution of an active agent; (c) forming particles; and (d)combining the diketopiperazine and the active agent; and thereafter (e)removing solvent by spray drying to obtain a dry powder, wherein the drypowder has an improved pharmaceutic property as compared to a dry powderobtained by removing solvent by lyophilization. In a further embodiment,the dry powder comprises an active agent such as insulin or an analoguethereof, but is not limited to such.

In still yet another particular embodiment, the present inventionprovides a method for delivering insulin to a patient in need thereof,comprising administering to the patient an effective amount of the drypowder.

The present invention also provides a dry powder having an improvedpharmaceutic property wherein the improved property is improved deliveryof the active agent whereby greater glucose disposal is achieved.

In still yet another particular embodiment of the present invention,there is provided a method of preparing a dry powder medicament with animproved pharmaceutic property, comprising: (a) providing adiketopiperazine in solution; (b) a step for forming particlescomprising the diketopiperazine; (c) and removing solvent by spraydrying to obtain a dry powder, wherein the dry powder has an improvedpharmaceutic property as compared to a dry powder obtained by removingsolvent by lyophilization. A further step comprising loading theparticle with an active agent prior to the solvent removal step is alsoprovided.

Another particular embodiment of the present invention provides a methodof optimizing the aerodynamic performance of a diketopiperazine drypowder comprising the steps of: (a) precipitating a diketopiperazinefrom solution under a controlled temperature to form particles; (b)selecting a drying method based on said temperature; and (c) drying theparticles. A further step comprising loading the particles with anactive agent is also contemplated.

In particular embodiments the inlet temperature during spray drying is105° C., 110° C., 120° C., 130° C., 140° C., or a range bounded by anypair of these values. In other particular embodiments the atomizationpressure is 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 bar or a rangebounded by any pair of these values. In further particular embodimentsthe spray rate is 4.4, 7.6, 12.2 g/min, or a range bounded by any pairof these values. In still another particular embodiment of the presentinvention the outlet temperature is 75° C.

In a further embodiment, the diketopiperazine is fumaryldiketopiperazine, wherein the controlled temperature is between about15° C. to about 18° C. and the selected drying method is spray drying.In other embodiments the controlled temperature is about 17° C. In stillother embodiments the controlled temperature is less than or equal toabout 13° C. or greater than or equal to about 19° C.

In a further particular embodiments there is contemplated a particlecontaining about 11.0%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%,11.8%, 11.9%, 12.0%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%,12.8%, 12.9%, 13.0%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%,13.8%, 13.9%, 14.0%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%,14.8%, 14.9%, 15.0%, 15.1%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%,15.9%, 16.0% or greater, insulin by weight. In a particular embodimentof the present invention there is provided a particle containing about11.4% insulin by weight. In another particular embodiment there iscontemplated a particle comprising up to 50% insulin by weight.

The active agent such as, but not limited to, insulin, comprised in asolution or suspension, is mixed with a suspension of a diketopiperazinewherein, the solution or suspension is in a suitable solvent for boththe active agent and the diketopiperazine

In some embodiments, the present invention provides a method ofobtaining a dry powder comprising a diketopiperazine and an active agentsuch as insulin, having improved pharmaceutic properties byprecipitating the particles from a solution at a controlled temperaturebetween about 15° C. to about 18° C. In other embodiments the controlledtemperature is about 17° C. In still other embodiments the controlledtemperature is less than or equal to about 13° C. or greater than orequal to about 19° C.

In other embodiments of the present invention the term ‘Cartridge FillWeight’ as used herein refers to the quantity of drug product containedin a cartridge for an inhaler, typically 5-10 mg or more. In otherembodiments the cartridge fill weight can vary from about 2.5 to 15 mg,10 to 20 mg, or 5 to 30 mg.

In further embodiments the bulk or tapped density of the powder dried byspray drying is increased compared to a similar powder dried bylyophilization. In one such embodiment the density is greater by afactor of about 2 (1.7-2.3). Particular further embodiments includethose limited to values disclosed in the examples or a range bounded byany pair of those values. In various embodiments the bulk density of thespray-dried powder is 0.150-0.200 g/cc. Particular embodiments includethose limited to values disclosed in the examples or a range bounded byany pair of those values. In various embodiments the tapped density ofthe spray-dried powder is 0.250-0.300 g/cc. Particular embodimentsinclude those limited to values disclosed in the examples or a rangebounded by any pair of those values.

In yet another embodiment of the present invention the term ‘CartridgeEmptying’ as used herein refers to the percentage (%) of powder that isdischarged from the inhaler upon activation (or discharge). This valueis typically obtained by weighing the cartridge before and afterdischarge. Particular embodiments include those limited to valuesdisclosed in the examples or a range bounded by any pair of thosevalues.

In still yet another embodiment of the present invention the term‘Respirable Fraction (RF)’ as used herein refers to the percentage (%)of particles in the respirable range (0.5-5.8 μm). The ‘RespirableFraction (RF) delivered’ refers to the percentage of active ingredientable to reach the airways of the lung where the pharmaceutical effect isexerted. Particular embodiments include those limited to valuesdisclosed in the examples or a range bounded by any pair of thosevalues.

In another embodiment of the present invention the term ‘RespirableFraction Based on Fill’ (′RF Based on Fill′, ‘% RF on Fill’ or‘°/0RF/fill’) as used herein refers to the percentage (%) of powder inthe respirable range normalized by the quantity of powder in theinhaler. Particular embodiments include those limited to valuesdisclosed in the examples or a range bounded by any pair of thosevalues.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present application and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 . Demonstration that increased atomization pressure had apositive effect on the aerodynamics of the diketopiperazine-insulinformulations. The inlet temperature ranged from 110° C. to 140° C. andthe outlet temperature was held constant at 75° C.

FIGS. 2A-2E. Evaluation of the accelerated stability ofdiketopiperazine-insulin formulations. The accelerated stabilityconditions were 40° C. and 75% RH (relative humidity) for 10 days. Areduction in insulin loss in the spray-dried formulations is depicted inFIG. 2A. FIG. 2B depicts a corresponding decrease in the formation ofA-21, the primary degradation product of insulin under these conditions.FIGS. 2C-2E demonstrate that the primary particles exhibit a decreasedtendency to aggregate as the atomization pressure is increased from 0.4bar (FIG. 2C) to 0.6 bar (FIG. 2D) to 0.7 bar (FIG. 2E). Themeasurements were obtained using laser diffraction.

FIG. 3 . Demonstration of the effect of temperature on the aerodynamicsof the diketopiperazine-insulin formulations. The outlet temperature washeld at 75° C. and the atomization pressure was held at 0.6 bar. The %RF on Fill (percent respirable fraction on a cartridge fill) remainedrelatively consistent over the temperature range.

FIGS. 4A-4F. Demonstration that increased inlet temperature (dryingrate) did not negatively impact the stability of the formulations. Theaccelerated stability conditions were 40° C. and 75% RH for 10 days.FIG. 4A depicts the percent loss of insulin. FIG. 4B depicts formationof A-21, the most prevalent degradation product.

FIG. 4C-4F depicts a trend towards increased aggregation of primaryparticles (as shown by the particles size distribution obtained fromlaser diffraction) as the inlet temperature is increased from 105° C.(FIG. 4C) to 120° C. (FIGS. 4D and 4E) to 140° C. (FIG. 4F).

FIGS. 5A-5E. Insulin distribution and particle morphology. FIG. 5A showsthat insulin is evenly distributed throughout the formulationindependent of particle size. FIGS. 5B-5E shows that the morphology ofthe spray-dried particles (FIGS. 5C and 5E) and lyophilized particles(FIGS. 5B and 5D) is the same.

FIGS. 6A-6B. Improvement in particle aerodynamics and insulin stability.FIG. 6A shows that % RF on Fill increases with atomization pressure at0.7, 0.9 and 1.1 bar respectively. FIG. 6B shows that % RF on Fill doesnot change with inlet temperature at 110° C., 120° C. and 130° C.respectively.

FIGS. 7A-7K. Demonstration that insulin stability increases at higherinlet temperatures and atomization pressures. FIG. 7A depictsmeasurement of the accelerated stability as percentage of insulin lossfor powders spray dried at a pressure of 0.7 bar and inlet temperaturesof 110° C., 120° C., and 130° C. respectively. FIG. 7B depictsmeasurement of the accelerated stability as percentage of insulin lossfor powders spray dried at a pressure of 1.1 bar and inlet temperaturesof 110° C., 120° C., and 130° C. respectively. FIGS. 7C-7K depictsminimal aggregation of primary particles (as shown by the particles sizedistribution obtained from laser diffraction) as the atomizationpressure was varied from 0.7-1.1 bar and the inlet temperature wasvaried from 110° C., 120° C., and 130° C. respectively.

FIG. 8 . Comparison of pharmacodynamic profiles (blood glucosereduction) following insufflation of 11.4% lyophilized FDKP/Insulin and11.4% spray dried FDKP/Insulin in rats. Each animal received 3 mg ofpowder containing 11.4% insulin by weight. Each group contained 4animals.

FIG. 9 . Aerodynamic performance of FDKP/Insulin powders dried by spraydrying or lyophilization. Two sets of suspensions (represented bysquares and circles) were tested. Opened symbols represent spray-driedpowders; filled symbols represent the lyophilized powders.

FIGS. 10A-10B. Stability data indicate that insulin loss (FIG. 10A) andA-21 formation (FIG. 10B) are reduced in the spray dried powder comparedto the lyophilized powder. Both powders were adjusted to pH 4.5 prior todrying.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

The success of any pharmaceutic particle depends not only on itsefficacy in treating a disease or condition, but also having superiorpharmaceutic properties over other known therapeutics. Desirablepharmaceutic properties sought in a dry powder particle include improvedaerodynamics, pharmacodynamics and stability. However, producingparticles with such properties is an ongoing challenge in the art. Oneapproach to achieving this aim in the art, lies in the methodology usedto manufacture particles.

Thus, the present invention provides the novel and unexpected discoverythat the pharmaceutic properties of the dry powder can be generallyimproved by using spray drying in preference to lyophilization to removesolvent from the particles.

The present invention serves to overcome the shortcomings in the art byproviding particles of a diketopiperazine (DKP) combined with an activeagent that are loaded and/or dried by a process to provide a dry powderhaving improved pharmaceutic properties. In particular embodiments, thepresent invention provides a particle, comprising a diketopiperazinecombined with insulin, dried by spray drying. The invention furtherprovides a spray-dried powder that demonstrates improved stability,aerodynamics or greater density, while maintaining at least similarpharmacodynamics as compared to the freeze-dried powder previouslydisclosed (see U.S. Pat. No. 6,444,226 entitled “Purification andStabilization of Peptide and Protein Pharmaceutical Agents” and U.S.patent application Serial Nos: 60/717,524, filed on Sep. 14, 2005 and11/532,063 filed Sep. 14, 2006, both entitled “Method of DrugFormulation Based on Increasing the Affinity of Active Agents forCrystalline Microparticle Surfaces”), each incorporated herein byreference for all they contain regarding diketopiperazine microparticlecompositions.

Diketopiperazine particles for drug delivery can be formed and loadedwith active agent by a variety of methods. Diketopiperazine solutionscan be mixed with solutions or suspensions of an active agent and thenprecipitated to form particles comprising the active agent.Alternatively the DKP can be precipitated to form particles andsubsequently mixed with a solution of the active agent. Associationbetween the particle and the active agent can occur spontaneously, bedriven by solvent removal, a specific step can be included prior todrying, or any combinations of these mechanisms applied to promote theassociation. Further variations along these lines will be apparent toone of skill in the art.

In one particular protocol the precipitated diketopiperazine particlesare washed, a solution of insulin is added, the mixture frozen bydropwise addition to liquid nitrogen and the resulting frozen droplets(pellets) lyophilized (freeze-dried) to obtain adiketopiperazine-insulin dry powder. In other embodiments, the mixturecan be dispersed into the liquid nitrogen by other means, for example,by spraying. In other protocols the precipitated diketopiperazineparticles of the invention are washed, a solution of insulin added, thepH of the solution adjusted to promote insulin adsorption onto theparticles, and solvent removed either by spray drying or freeze dryingto obtain a diketopiperazine-insulin dry powder. Previously,lyophilization had been used for solvent removal and it had beenexpected that the use of spray drying for this purpose would producesimilar results. As disclosed herein, it was surprisingly discoveredthat spray-dried dry powder possessed improved pharmaceuticcharacteristics. In particular the spray-dried powder had an improvedrespirable fraction (% RF), the insulin contained in the particles hadgreater stability against degradation and the particles had a greaterdensity allowing higher doses to be loaded into any particular volume.Upon pulmonary administration, at least comparable amounts of insulinwere delivered into the bloodstream as evidenced by at least comparablereductions in blood glucose. The performance of the spray-dried powderswas superior to the lyophilized powders whether or not the preparationof the lyophilized samples included a pH-adjustment to promoteassociation of the drug with the particle.

In a further refinement of the methodology, the temperature of thesolution from which the DKP was precipitated was controlled.Surprisingly, FDKP particles precipitated from solutions at temperaturesabout 13° C. or about 9° C., dry powders with greater % RF were obtainedusing lyophilization for solvent removal. For FDKP particlesprecipitated from solutions at temperatures at about 17° C., dry powderswith greater % RF were obtained using spray drying for solvent removal.In the remaining portions of the tested range, aerodynamic performancewas similar with either drying method. Thus aerodynamic performance ofDKP particles can be optimized by selecting a solvent removal procedureon the basis of the temperature of the solution from which the particlesare precipitated. The dry powders obtained were characterized foraerodynamic properties (% RF, cartridge emptying, % RF/fill, mass medianaerodynamic diameter [MMAD], geometric standard deviation [GSD]) andphysicochemical properties (insulin content [(% load], yield, density)as described in examples provided herein.

Surprisingly, the density of the spray-dried particles was roughly twicethat of freeze-dried particles. This can be advantageous in providinghigher doses. Dry powder inhalers generally impose a limit on the volumeof powder, and thus the dosage of active agent, that can be delivered ina single operation. A powder of higher density, but at least similarrespirable fraction, allows larger doses to be administered in a singleoperation, rather than requiring more operations per dose, formulationswith higher % loading of active agent, or alternate inhaler or inhalercartridge designs to accommodate various volumes of powder. Any of thesealternatives entail greater development and/or production costs and alsointroduce issues of product complexity. Product complexity andrequirements for multiple operations per dose additionally create issueswith product acceptance and patient compliance. Thus this unexpectedincrease in powder density offers multiple advantages for the use ofspray-dried powders as pharmaceutical products.

1. Preparing Preformed Particles by Spray Drying

Spray drying, as employed in the present invention, is a thermalprocessing method used to load and/or dry particles in a suspension in aliquid medium (solvent). As disclosed in the examples herein, asuspension of diketopiperazine particles and an insulin solution aremixed. Some or all of the insulin molecules then bind to thediketopiperazine particles. In various embodiments thediketopiperazine-insulin particles are then loaded and/or dried by spraydrying and a dry powder is obtained. In an alternative embodiment, theactive agent is added to a diketopiperazine solution prior toprecipitation of the particles.

During spray drying, the aqueous mixture of diketopiperazine particlesor diketopiperazine-insulin particles, are introduced via a nozzle(e.g., a two fluid nozzle or high pressure nozzle), spinning disc, or anequivalent device into a heated gas stream. Prior to being passedthrough the heated gas stream, the solution or suspension is atomizedinto fine droplets. The heat energy supplied by the gas stream causesthe evaporation of water and other solvents in which the particles aresuspended, thereby producing dry powder compositions.

In obtaining a dry powder comprising a diketopiperazine combined withinsulin, as in embodiments of the present invention, the inventors foundthat the spray drying method generally provided particles with superiorpharmaceutic properties compared to similar particles obtained by freezedrying. In obtaining the particles, the inventors took intoconsideration a number of parameters. These parameters includedtemperature, atomization pressure, solids content of the suspensions,percent of insulin loss, formation of A-21, aggregation of particles,and aerodynamic and biological performance.

The inlet temperature is the temperature of the gas stream leaving itssource. The outlet temperature is a measure of the final temperature ofthe powder formulation and an indication of the utilization of theenergy in the inlet air for drying and is a function of the inlettemperature and the heat load required to dry the product, along withother factors. The outlet temperature is selected based upon thelability of the macromolecule being treated.

The diketopiperazine/active agent mixture may be a suspension. Thesolvent, generally water, rapidly evaporates from the droplets producinga fine dry powder.

Spray drying is performed under conditions that result in a powder ofhomogeneous constitution having a particle size that is respirable, withlow moisture content and other characteristics that allow foraerosolization. Preferably the particle size of the resulting powder issuch that more than about 98% of the particles (by mass) have a diameterof about 10 μm or less with about 90% of the particles (by mass) have adiameter less than 5 μm. Alternatively, about 95% of the particles (bymass) have a diameter of less than 10 μm with about 80% of the particles(by mass) have a diameter of less than 5 μm. In certain embodiments, thedry powder has a mean particle size of 1 to 5 μm in diameter. Thepreceding embodiments relate especially to use of the powder inpulmonary delivery. Mean particle size can effect where in therespiratory tract particles are deposited and can also effect their bulkhandling properties. For example nasal deposition is favored forparticles with mean diameters greater than 20 μm. In other embodiments,the powder may be used to form tablets, packaged in capsules, orresuspended for oral administration or injection. Thus in variousembodiments, the dry powder may comprise particles having a meanparticle size of greater than about 10 μm, 20 μm, 30 μm, 40 μm, 50 μm,60 μm, 70 μm, 80 μm, 90 μm, 100 μm. In another embodiment, the drypowder may comprise particles having a mean particle size of about 100μm to about 500 μm. In other embodiments, the dry powder may compriseparticles having a mean particle size of less than about 1 mm.

Suspensions of the present invention, comprising an active agent and adiketopiperazine may be spray-dried in conventional spray dryingequipment such as the PHARMASD™ PSD-1 Spray Dryer or the SD-Micro™ SprayDryer, as are well known in the art and obtainable from a commercialsupplier (Niro Inc., Columbia, MD), thereby resulting in a dry powdercomprised of such particles. It is noted that other conventional spraydrying equipment may be used.

In conducting spray drying experimentation, methods such as rotaryatomization, pressure atomization, and two-fluid atomization (forexample, co-current two-fluid nozzle and/or fountain two-fluid nozzle)may be employed. Devices used in spray drying methodology are well knownto one of ordinary skill in the art.

Although no special restrictions are placed on the nozzle of theatomizer used in the process of spraying, for a nozzle which can producea spray-dry composition with a grain diameter suitable for nasalpharyngeal or pulmonary administration it is recommended in the art touse nozzles such as those in the following examples. For example, nozzletypes “1A,” “1,” “2A,” “2,” “3” and the like, (manufactured by YamatoChemical Co.), or the SB Series SprayDry® Nozzles (manufactured bySpraying Systems Co.), can be used with the spray-dryer. In addition,disks type “MC-50,” “MC-65” or “MC-85,” (manufactured by Okawara KakokiCo.), can be used as rotary disks of the spray-drier atomizer.

In other embodiments, the inlet gas temperature used to dry the sprayedmaterial is such that it does not cause heat deactivation of the activeagent. The range of inlet temperatures may vary between about 50° C. toabout 200° C., preferably between about 110° C. and 160° C. Withwell-stabilized agents, the inlet temperature can exceed 200° C. Thetemperature of the outlet gas used to dry the sprayed material may varybetween about 35° C. and about 100° C., preferably between 55° C. and85° C. In other embodiments, the outlet temperature may be preferably at75° C. In another embodiment of the present invention, the inlet andoutlet temperatures may be held at 120° C. and 75° C. respectively.

As disclosed above and elsewhere herein, terminology useful andapplicable to the methods and compositions of the present invention areas follows:

-   -   The term “powder” means a composition that consists of fine        solid particles that are capable of being dispersed in an        inhalation device and inhaled by a subject. In preferred        embodiments the particles reach the lungs or alveoli. Such a        powder is said to be “respirable.” Preferably the average        particle size is less than about 10 microns (μm) in diameter        with a relatively uniform spheroidal shape distribution. More        preferably the diameter is less than about 7.5 μm and most        preferably less than about 5.0 μm. Usually, the particle size        distribution is between about 0.1 μm and about 8 μm in diameter,        particularly about 0.3 μm to about 5 μm.

The term “dry” means that the powder composition is not suspended ordissolved in a propellant, carrier, or other liquid. It is not meant toimply a complete absence of water. The composition can have a moisturecontent such that the particles are readily dispersible in an inhalationdevice to form an aerosol. This moisture content is generally belowabout 10% by weight (% w) water, usually below about 5% weight andpreferably less than about 3% weight.

The term “effective amount” is the amount that is needed to provide adesired response in the subject to be treated. The precise dosage willvary according to a variety of factors including, but not limited to,the age and size of the subject, the disease and the treatment beingeffected. The “effective amount” will also be determined based on theanticipated pharmacodynamic response or bioavailability.

2. Diketopiperazines

Diketopiperazines can be formed into particles that incorporate anactive agent or particles onto which an active agent can be adsorbed.Diketopiperazines of the present invention include but are not limited3,6-di(fumaryl-4 aminobutyl)-2,5-diketopiperazine also known as(E)-3,6-bis[4-(N-carboxyl-2-propenyl)amidobutyl]-2,5-diketopiperazine(which may also be referred to as fumaryl diketopiperazine or FDKP).

Other diketopiperazines that are contemplated in the present inventioninclude 3,6-di(4-aminobutyl)-2,5-diketopiperazine;3,6-di(succinyl-4-aminobutyl)-2,5-diketopiperazine (succinyldiketopiperazine or SDKP);3,6-di(maleyl-4-aminobutyl)-2,5-diketopiperazine;3,6-di(citraconyl-4-aminobutyl)-2-5-diketopiperazine;3,6-di(glutaryl-4-aminobutyl)-2,5-diketopiperazine;3,6-di(malonyl-4-aminobutyl)-2,5-diketopiperazine;3,6-di(oxalyl-4-aminobutyl)-2,5-diketopiperazine and derivativestherefrom.

In brevity, diketopiperazines can be formed by cyclodimerization ofamino acid ester derivatives, as described by Katchalski, et al., (J.Amer. Chem. Soc. 68:879-80; 1946), by cyclization of dipeptide esterderivatives, or by thermal dehydration of amino acid derivatives inhigh-boiling solvents, as described by Kopple, et al., (J. Org. Chem.33(2):862-64;1968), the teachings of which are incorporated herein.

Methods for synthesis and preparation of diketopiperazines are wellknown to one of ordinary skill in the art and are disclosed in U.S. Pat.Nos. 5,352,461; 5,503,852; 6,071,497; 6,331,318; and 6,428,771; and U.S.patent application Ser. No. 11/208,087 each of which is incorporatedherein by reference for all they teach regarding diketopiperazines. U.S.Pat. No. 6,444,226, herein incorporated by reference for all it containsregarding diketopiperazine microparticles, describes preparing andproviding microparticles of diketopiperazines in aqueous suspension towhich a solution of active agent is added. This patent further describesa method of removing a liquid medium by lyophilization to yieldmicroparticles comprising an active agent. See also U.S. Pat. No.6,440,463 and U.S. patent application Serial Nos: 11/532,063 and11/532,025 both filed on Sep. 14, 2006, and U.S. Provisional patentapplication Ser. No. 60/717,524, filed on Sep. 14, 2005; each of whichis incorporated herein by reference for all they teach regardingdiketopiperazine microparticles.

In one embodiment, active agent is encapsulated within microparticles bydissolving a diketopiperazine with acidic side chains in bicarbonate orother basic solution, adding the active agent in solution or suspension,and then precipitating the microparticle by adding acid, such as 1 Mcitric acid. In another embodiment, active agent is encapsulated withinmicroparticles by dissolving a diketopiperazine with basic side chainsin an acidic solution, such as 1 M citric acid, adding the active agentin solution or suspension, and then precipitating the microparticle byadding bicarbonate or another basic solution. In still anotherembodiment, active agent is encapsulated within microparticles bydissolving a diketopiperazine with both acidic and basic side chains inan acidic or basic solution, adding the active agent in solution orsuspension to be encapsulated, then precipitating the microparticle byneutralizing the solution. In an alternative embodiment, microparticlesof diketopiperazine are prepared and provided in a suspension, typicallyan aqueous suspension, to which a solution of the active agent then isadded.

It is further contemplated that the diketopiperazine-insulin particleformulations of the present invention can be administered by variousroutes of administration. As dry powders these particles can bedelivered by inhalation to specific areas of the respiratory system,depending on particle size. Additionally, the particles can be madesmall enough for incorporation into an intravenous suspension dosageform. Oral delivery is also possible with the particles incorporatedinto a suspension, tablets, or capsules.

3. Active Agents

Embodiments of the present invention employ particles combining anactive agent with a diketopiperazine. The term ‘active agent’ isreferred to herein as the therapeutic agent, or molecule (such asprotein or peptide or biological molecule), to be encapsulated,associated, joined, complexed or entrapped in or to the diketopiperazineof the present invention. Generally speaking, any form of an activeagent can be combined with a diketopiperazine of the present invention.Active agents, as contemplated in the present invention, may or may notbe charged.

Active agents contemplated for use in the compositions and methodsdescribed herein may include any polymer or large organic molecules,most preferably peptides and proteins. Examples include syntheticorganic compounds, proteins and peptides, polysaccharides and othersugars, lipids, and nucleic acid sequences, having therapeutic,prophylactic, or diagnostic activities. Active agents may also includesmall molecules and vitamins. An active agent of the present inventionmay also be a vasoactive agent, a neuroactive agent, a hormone, an agentregulating metabolism, weight, or blood glucose levels, ananticoagulant, an immunomodulating agent, a cytotoxic agent, anantibiotic, an antiviral, an antisense molecule, or an antibody.

Examples of specific exemplary active agents have been listed above. Inparticular embodiments of the invention the active agent is insulin oran analogue thereof. Analogues with faster, slower, shorter, or longeraction profiles are known in the art. Such analogues include those withaltered amino acid sequences and those that have been covalentlymodified with other moieties, such as polyethylene glycol, or additionalamino acids, such as in a fusion protein. Ultimately any molecule with asubstantial portion of a wild type insulin molecule and physiologicallyrelevant insulin activity is comprehended by this term.

Proteins as contemplated by the present invention are defined asconsisting of 100 amino acid residues or more; in addition, peptidescontemplated by the invention are less than 100 amino acid residues.

4. Stabilizing Agents Contemplated in the Present Invention

In further embodiments, there is contemplated by the present inventionthe use of stabilizing agents that may be contained in a suspension orsolution comprising a diketopiperazine and an active agent which may beincorporated into the particle formulation.

Stabilizing agents may be included for conformational stability duringthe drying process. In addition, these stabilizing agents may furtherimprove the aerodynamics or bioavailability of the dry powderdiketopiperazine-insulin particle formulations of the present invention.Such stabilizing agents may comprise, but are not limited to, sugars,surface modifying agents, surfactants, hydrophobic amino acids such astryptophan, tyrosine, leucine, phenylalanine, pharmaceutical carriers orexcipients, and the like.

Stabilizing agents contemplated by the present invention are thosepreferably suitable for respiratory and pulmonary administration. Incertain embodiments, it is preferred that the stabilizing agent beincorporated simultaneously into the diketopiperazine-insulin particleto produce a homogeneous powder. Alternatively, the stabilizing agentmay be separately prepared in a dry powder form and combined with thespray dried diketopiperazine-insulin particle by blending.

In other instances, powder carriers may be employed such as, but notlimited to carbohydrates, e.g., monosaccharides such as fructose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, trehalose, cellobiose, and the like; cyclodextrins,2-hydroxypropyl-β-cyclodextrin; and polysaccharides, such as raffinose,maltodextrins, dextrans, and the like; amino acids, such as glycine,arginine, aspartic acid, glutamic acid, cysteine, lysine, and the like;organic salts prepared from organic acids and bases, such as sodiumcitrate, sodium ascorbate, magnesium gluconate, sodium gluconate,tromethamine hydrochloride, and the like; peptides and proteins, such asaspartame, human serum albumin, gelatin, and the like; alditols, such asxylitol, and the like. A preferred group of carriers may includetrehalose, raffinose, maltodextrins, glycine, sodium citrate,tromethamine hydrochloride, human serum albumin, and mannitol. Suchpowder carriers will usually be crystalline (to avoid water absorption),but might in some cases be amorphous or mixtures of crystalline andamorphous forms. The size of the stabilizing agent particles may beselected to improve the flowability of the spray dried powder product.

Sugars as contemplated by the present invention include, but are notlimited to, dextrose, lactose, and mannitol.

Surfactants as contemplated by the present invention include, but arenot limited to, polysorbate 80 (PS80), lecithin, phosphatidylcholine,DPPC, sodium dodecylsulfate, and ionic detergents.

5. Method for Coating Microparticles

The procedure for coating crystalline microparticles, such as preformedcrystalline microparticles, with active agents is described generally asfollows: crystalline microparticles previously formed by precipitation,or another method, are suspended in liquid medium, such as water; andthe medium is adjusted to alter the particles' surface either before orafter addition of active agent. At this point the active agent willadsorb to the microparticle surface and after an interval of time (forexample <1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes; preferably from <1to at least 5 minutes) the loading process will be complete. The liquidmedium may then be removed by spray-drying.

6. Promoting Adsorption of Active Agents

Adsorbing active agent to the surface of a crystalline microparticle caninvolve altering the properties of the active agent in a solution orfluid suspension under various solution conditions, thereby promotingadsorption to the microparticle surface and reducing the amount ofactive agent remaining in solution. Alteration or modifications to theactive agent may occur with the use of modifiers such as, but notlimited to, chaotropes and kosmotropes, salts, organics such as, but notlimited to, alcohols, osmolytes, and surfactants. These modifiers canact on the active agent to alter its chemical potential and thereby itsstructure, flexibility, rigidity or stability, without chemicallyaltering the agent itself. The term “chemical potential” is well knownto one of ordinary skill. In embodiments of the present invention,“chemical potential” refers to the free energy necessary to drive achemical reaction such as, for example, interaction between an activeagent and a solvent or the adsorption of active agent onto amicroparticle. The term “energetically favorable” as used herein refersto the lowering of the free energy levels of the adsorbed states of theactive agent onto the microparticle in relation to the free energy levelof uncoated microparticle, or unbound active agent and/or the insolubleforms (including aggregation or precipitation) of the active agent. Theterm “structure” as used herein refers to the secondary structure of theactive agent molecule and includes the alpha-helical formation, betasheets, or random coil (unordered) of the active agent molecule, such asa protein. Additionally, the term structure may also include teritaryand quaternary structures of the molecule but is not limited to such andmay also refer to the self association, aggregation, multimerization,dimerization, and the like, of a molecule. The term “stability” as usedherein refers to the stabilization or destabilization of the structureof the active agent in the presence of the modifier.

In addition, altering the properties of the active agent in a solutionor fluid suspension are likely to affect the interactions due tohydrophobic properties, hydrogen bonding properties, and electrostaticproperties of the active agent and/or microparticle.

Hydrophobic interactions are associations of non-polar groups with eachother in aqueous solutions because of their insolubility in water.Hydrophobic interactions can affect a number of molecular processesincluding, but not limited to, structure stabilization (of singlemolecules, complexes of two or three molecules, or larger assemblies)and dynamics, and make important contributions to protein-protein andprotein-ligand binding processes. These interactions are also known toplay a role in early events of protein folding, and are involved incomplex assembly and self-assembly phenomena (e.g., formation ofmembranes).

Hydrogen bonding interactions are especially strong dipole-dipole forcesbetween molecules; a hydrogen atom in a polar bond (e.g., H—F, H—O orH—N) can experience an attractive force with a neighboringelectronegative molecule or ion, which has an unshared pair of electrons(typically an F, O, or N atom on another molecule). Hydrogen bonds areresponsible for the unique properties of water and are very important inthe organization of biological molecules, especially in influencing thestructure of proteins and DNA.

Electrostatic interactions are attractions between opposite charges orrepulsions between like charges that grow stronger as the charges comecloser to each other. Electrostatic interactions constitute a keycomponent in understanding interactions between charged bodies in ionicsolutions. For example, the stability of colloidal particles dispersedin a solvent can be explained by considering the competition betweenrepulsive electrostatic interactions and the attractive van der Waalsinteractions. Electrostatic interactions are also of importance whenconsidering interaction and adhesion between particles.

7. Manipulating Microparticle Surface Properties

As disclosed elsewhere herein, the surface properties of themicroparticle can be manipulated by various means. The microparticlesurface properties that can be manipulated include, but are not limitedto, electrostatic, hydrophobic, and hydrogen bonding properties. Invarious embodiments these manipulations are carried out in the absenceor presence of the active agent, or before or after the microparticlesand the active agent are mixed together. When the manipulation takesplace in the presence of the active agent, for example by alteringsolution condition, there can also be effects on the active agent thatwill modify its affinity for the surface. Thus in some embodiments ofthe present invention, coating of the microparticle can involvemanipulation of surface properties and modification of properties of theactive agent. Methods directed to the latter are disclosed in U.S. Pat.No. 7,803,404 entitled METHOD OF DRUG FORMULATION BASED ON INCREASINGTHE AFFINITY OF ACTIVE AGENTS FOR CRYSTALLINE MICROPARTICLE SURFACES andwhich is incorporated herein by reference in its entirety.

Electrostatic interactions are attractions between opposite charges orrepulsions between like charges that grow stronger as the charges comecloser to each other. Electrostatic interactions constitute a keycomponent in understanding interactions between charged bodies in ionicsolutions. For example, the stability of colloidal particles dispersedin a solvent can be explained by considering the competition betweenrepulsive electrostatic interactions and the attractive van der Waalsinteractions. Also, the chemical functionality (for example, but notlimited to COOH, NH, etc.) of the microparticle surface can be utilizedas the counter ion to an ionized active agent such that the activeagent/particle composite comprises a salt. Electrostatic interactionsare also of importance when considering interaction and adhesion betweenparticles.

Altering the pH of the surrounding solution system can changeelectrostatic properties of ionizable crystalline microparticles insuspension. Changing the pH of the solution changes the ionization of amicroparticle such that active agent adsorbs to the microparticlesurface. Microparticles composed of FDKP(3,6-bis[N-fumaryl-N-(n-butyl)amino]2,5-diketopiperazine) are ionizable.The microparticles are insoluble in water below pH 3.5 but solubilityincreases rapidly between pH 3.5 and 5.0, presumably due to theionization of the carboxyl groups. The FDKP microparticle is partiallyionized at pH 5 prior to complete dissolution at higher pH, which can beobserved indirectly via ultrasonic spectroscopy. In one embodiment,diketopiperazine microparticles are suspended in an acidic solution,active agent is added to the suspension, and the pH of the solution israised after the active agent and microparticles are mixed together. Theincreased pH alters the surface properties of the microparticles tocreate an environment in which the active agent has a higher affinityfor the microparticle than for the solvent.

Alternatively, the pH of the microparticle suspension can be raisedimmediately prior to addition of active agent to the solution. Thesurface charge properties of the microparticle are altered by the changein pH such that active agent has a higher affinity for the microparticlethan for remaining in solution and is adsorbed to the microparticlesurface upon addition.

8. Other Methods of Manipulating the Surfaces of Microparticles

In addition to electrostatic properties, other properties of amicroparticle surface can be exploited to control adsorption of activeagent. Microparticles containing compounds with imidazole, pyridine,Schiff bases, ketone, carboxylic acid bioisosteres, amides, or otherfunctional groups that can exist in multiple structures could bemanipulated to modify surface properties.

Hydrophobic interactions are associations of non-polar groups with eachother in aqueous solutions because of their insolubility in water.Hydrophobic interactions can affect a number of molecular processesincluding, but not limited to, structure stabilization (be it of singlemolecules, complexes of two or three molecules, or larger assemblies)and dynamics, and make important contributions to protein-protein andprotein-ligand binding processes. These interactions are also known toplay a role in early events of protein folding, and are involved incomplex assembly and self-assembly phenomena (e.g., formation ofmembranes).

Hydrophobic interactions can be manipulated by changing the protonationof crystalline microparticles composed of histidine. Protonating thehistidine will reduce the nucelophilicity of the crystallinemicroparticles and impart a positive charge.

Hydrogen bonding interactions are especially strong dipole-dipole forcesbetween molecules; a hydrogen atom in a polar bond (e.g., H—F, H—O orH—N) can experience an attractive force with a neighboringelectronegative molecule or ion, which has an unshared pair of electrons(usually an F, O or N atom on another molecule). Hydrogen bonds areresponsible for the unique properties of water and are very important inthe organization of biological molecules, especially in influencing thestructure of proteins and DNA.

In the present invention, the hydrogen bonding properties of themicroparticle surface can be controlled by chemical derivatization.Hydrogen bond donors/acceptors can be added chemically to alter themicroparticle surface. For example, the hydrogen in an N—H bond canundergo hydrogen bonding to the oxygen in a C═O bond. If the N—H isreplaced by an N—CH₃, then this particular hydrogen bonding interactionis removed. Likewise, replacement of the C═O group with a C═C group alsoremoves this particular bonding interaction.

Microparticles with surfaces containing ionizable aromatic groups arepolar when ionized but hydrophobic in their un-ionized state. Startingwith protonated surfaces and manipulating solution conditions to reduceparticle surface ionization causes hydrophobic or aromatic active agentsto coat the microparticle surface.

Microparticles with ketone surface groups could be manipulated bychanging the solution polarity. By reducing solvent polarity (adding lowpolarity organic solvents to an aqueous solution) the enol-form is madethe predominant species at the particle surface. This enol-form is ahydrogen bond donor whereas the keto-form is a hydrogen bond acceptor.The adsorption of nitrogen-containing drugs onto the microparticlesurface is promoted in this manner.

Microparticles with surface groups that undergo pH- ortemperature-induced isomerization can also be induced to adsorb drugmolecules by manipulating solution conditions. In the case of thesesurfaces, the introduction of a kink in a linear surface group due toisomerization increases the mobility (fluidity) of the groups at themicroparticle surface. This allows the surface to form more contactswith the active agent than are possible with an ordered surface. If theadditional interactions with the active agent are each favorable, thenthe net interaction energy becomes favorable and the drug adsorbs to themicroparticle surface.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Effect of Atomization Pressure on Aerodynamics, Stability, andAggregation

Diketopiperazine derivative,3,6-bis[N-fumaryl-N-(n-butyl)amino]-2,5-diketopiperazine (also referredto as 3,6-di(fumaryl-4 aminobutyl)-2,5-diketopiperazine, fumaryldiketopiperazine or FDKP; also termed(E)-3,6-bis[4-(N-carboxy-2-propenyl)amidobutyl]-2,5-diketopiperazine)were precipitated and washed. Insulin was loaded onto the FDKP particlesby adjustment to a pH of approximately 4.45, and the FDKP-insulinparticles were spray dried to obtain a FDKP-insulin dry powder. A pH ofabout 4.45 was found to increase the binding of insulin to FDKPparticles as disclosed in U.S. patent application Serial Nos: 11/532,063and 11/532,025 both filed on Sep. 14, 2006, and U.S. Provisional patentapplication Ser. No. 60/717,524, filed on Sep. 14, 2005.

The dry powders were characterized for various aerodynamic properties (%RF, cartridge emptying, % RF/fill, mass median aerodynamic diameter[MMAD], and geometric standard deviation [GSD]).

Table 1 and FIG. 1 demonstrate the effect of the atomization (nozzle)pressure on the aerodynamic performance of the particles. The nozzlepressures ranged from 0.4 bar to 1.1 bar (Table 1). The respirablefraction on fill (% RF on Fill) improved as the atomization pressure wasincreased from 0.4 bar to 1.1 bar.

TABLE 1 Effect of atomization pressure on aerodynamic properties. Outlettemperature was 75° C. Atomization % Pressure Inlet T % RF Cartridge %RF MMAD (bar) (° C.) delivered Emptying fill (μm) GSD 0.4 105 34.7 95.533.1 2.7 2.2 0.5 105 30.3 92.1 27.9 3.3 2.3 0.6 105 39.4 95.6 37.7 2.52.3 120 45.5 91.9 41.8 2.7 2.2 120 45.4 92.2 41.9 2.5 2.2 140 42.4 91.438.8 2.5 2.2 0.7 105 48.2 92.7 44.7 2.7 2.2 110 71.9 68.9 49.5 2.3 2.0120 57.7 77.6 44.8 2.5 2.0 130 63.5 71.6 45.5 1.9 2.0 0.9 110 68.4 70.248.0 2.3 2.0 120 68.3 74.9 51.2 2.1 2.0 130 55.4 90.2 49.9 2.7 2.0 1.1110 64.2 84.0 54.0 2.5 1.9 120 70.4 70.8 49.8 2.0 2.0 130 71.7 74.9 53.72.2 2.0

The stability of the insulin was assessed as the percent loss of insulin(FIG. 2A) and the percent conversion to insulin degradation productA21-desamido insulin (% A21) under stress conditions (10 days at 40° C.,75% RH) (see FIG. 2B). For comparison, the bottom bar in each figurerepresents data obtained with freeze dried particles. The datademonstrate that, as the atomization pressure was increased, there was ageneral trend toward increased stability of the insulin in thediketopiperazine-insulin particles. Less formation of the A21 insulindegradation product was observed in all of the spray-dried particles ascompared to freeze-dried particles (FIG. 2B).

To assess aggregation of the primary particles, the particle sizedistribution was determined from laser diffraction of a suspension ofspray-dried particles using a Malvern Mastersizer 2000. Under the abovetrial conditions, a trend towards decreased aggregation of the primarydiketopiperazine-insulin particles was observed with increasedatomization pressure (FIGS. 2C-2E). It is observed that the size of thepeak to the right, representing aggregated particles, decreases as theatomization pressure increases from 0.4 bar (FIG. 2C) to 0.6 bar (FIG.2D), to 0.7 bar (FIG. 2E).

Example 2 Effect of Inlet Temperatures on Aerodynamics, Stability, andParticle Aggregation

Using particles prepared as above, spray dryer inlet temperature andprocess scalability were evaluated as shown in Table 2 below. In theseexperiments, the inlet temperature was varied from 105° C. to 140° C.and the outlet temperature was held constant at 75° C. The nozzlepressure was held constant at 0.6 bar.

It was observed that the increased inlet temperatures required anincrease in the spray rate to maintain a consistent outlet temperature(Table 2). The increased spray rates produced dried particles at agreater production rate. The aerodynamics of the spray dried particleswere assessed (Table 2). The % RF on Fill remained consistent over thetemperature range studied (FIG. 3 ).

TABLE 2 Effect of inlet temperature on particle aerodynamics. Nozzlepressure was maintained at 0.6 bar and the outlet temperature was 75° C.Spray Inlet % Scale Rate Temp. cartridge % RF MMAD (g) (g/min) (° C.) %RF emptying on Fill (μm) GSD 11.3 4.4 105 39.4 95.6 37.7 2.5 2.3 11.37.6 120 45.5 91.9 41.8 2.7 2.2 45.2 7.6 120 42.4 91.4 38.8 2.5 2.2 11.312.2 140 45.4 92.2 41.9 2.5 2.2

Further, the data demonstrated that increasing the inlet temperature(drying rate) did not negatively impact the stability of the insulin onthe particles. There was a trend toward increased insulin stability withincreasing inlet temperature. Stability was measured as insulin lost andA21 formed (FIGS. 4A and 4B) after 10 days at However, under the abovetrial conditions, a trend toward increase aggregation of the primarydiketopiperazine-insulin particles was observed with an increase in theinlet temperature (FIGS. 4C-4F).

Example 3 Insulin Recovery and Distribution

In these experiments, a known mass of diketopiperazine particles wassuspended in water. Enough insulin solution of known concentration wasadded to the suspension to give a theoretical composition of 11.4%insulin. The fumaryl diketopiperazine-insulin slurry was titrated to apH of approximately 4.45 prior to spray drying.

Insulin distribution across particles was assessed as shown in FIG. 5A.These experiments were conducted using an Andersen Cascade Impactor. Thepowder was filled into cartridges and discharged through a MedTone®inhaler into the Andersen cascade impactor. (The MedTone® inhaler isdescribed in U.S. patent application No. 10/655,153 entitled “Unit DoseCartridge and Dry Powder Inhaler” which is incorporated herein byreference for all it contains regarding the inhaler device). Theimpactor classifies the particles by aerodynamic size. After discharge,the powder was recovered from each stage and assayed for insulin content(load). Insulin is shown to be evenly distributed throughout theformulation. Increasing the scale (grams of the powder), as shown inTable 2 above, by a factor of 4 was also found to be acceptable.

Particle morphology of the spray-dried and lyophilized particles wascompared by scanning electron microscopy (SEM). FIG. 5B-5E shows theparticle morphologies for the lyophilized formulation (FIGS. 5B and 5D)are comparable to those for the spray-dried formulation (FIGS. 5C and5E).

Summary of Examples 1-3

The above data, show that: 1) increasing the atomization pressuredecreased the aggregation of the primary particles; 2) increasing theinlet temperature had little impact on the particles aerodynamics; 3)increasing the inlet temperature was not observed to have a negativeimpact on the stability of the insulin; 4) increasing the inlettemperature resulted in greater aggregation of the primary particles; 5)spray-dried particles had increased insulin stability when compared tolyophilized particles of identical composition; and 6) spray-driedparticles had similar morphology as lyophilized particles.

Example 4 Determination of Spray-drying Parameters to MaximizeAerodynamic Performance

Inlet temperature and atomization pressure were further evaluated usinginlet temperatures of 110, 120 and 130° C. and atomization pressures of0.7, 0.9, and 1.1 bar (Table 3).

TABLE 3 Effect of spray-drying parameters on particle aerodynamicsAtomization Inlet % pressure Temperature Cartridge % RF MMAD (bar) (°C.) % RF emptying on fill (μm) GSD 0.7 110 71.9 68.9 49.5 2.3 2.0 0.7120 57.7 77.6 44.8 2.5 2.0 0.7 130 63.5 71.6 45.5 1.9 2.0 0.9 110 68.470.2 48.0 2.3 2.0 0.9 120 68.3 74.9 51.2 2.1 2.0 0.9 130 55.4 90.2 49.92.7 2.0 1.1 110 64.2 84.0 54.0 2.5 1.9 1.1 120 70.4 70.8 49.8 2.0 2.01.1 130 71.7 74.9 53.7 2.2 2.0

FIG. 6A summarizes the results of Table 3 as the % RF on fill versus theatomization pressure; FIG. 6B summarizes the results as % RF on fillversus the inlet temperature. Thus, the data show that increasingatomization pressure leads to improved aerodynamic performance and inlettemperature does not affect this parameter.

Example 5 Effect of Inlet Temperature and Atomization Pressure onStability and Aggregation

The samples listed in Table 3 were analyzed for insulin stability andparticle aggregation. As shown in FIGS. 7A and 7B, the results wereconsistent with those of Examples 1-3 in that the spray-dried samplesshowed less insulin loss than comparable lyophilized powders (the bottombar in FIGS. 7A and 7B; loading of the particles used in the lyophilizedsamples included adjustment to pH 4.5, which as discussed in Example 1above, increases the binding of insulin to FDKP particles).

The aggregation of the primary diketopiperazine-insulin particle wasassessed under the conditions of increased inlet temperature andincreased atomization pressure (FIGS. 7C-7K) The particle sizedistributions by laser diffraction were generally insensitive toatomization pressure and temperature over the ranges covered in thisexample. A small degree of aggregation was observed at 0.7 bar and inlettemperatures of 110° C. and 120° C., but a unimodal distribution wasobtained at all other conditions.

The results for the spray-dried samples as compared to the lyophilizedsamples show: 1) the atomization pressure can be increased to improveaerodynamics; 2) the inlet temperature has negligible effect on % RF onFill; 3) insulin stability increases with increased inlet temperature;and 4) the increased inlet temperature and atomization pressure reducedaggregation of the primary insulin particles.

Example 6 Insulin Pharmacodynamics with Spray-dried Particles

Data from a rat insufflation study indicated that spray-driedFDKP-insulin powder provides at least comparable glucose disposal asprovided by lyophilized material. FIG. 8 shows a comparison ofpharmacodynamic profiles (blood glucose reduction) followinginsufflation of lyophilized and spray-dried 11.4% FDKP-insulin particlesin rats. The glucose lowering capacity of spray-dried FDKP-insulinpowder was found to be equivalent to that of lyophilized FDKP-insulinpowder.

Example 7 Aerodynamics and Stability of Spray-dried FDKP-insulin Powder

Fumaryl diketopiperazine (FDKP)-insulin particles were prepared in amanner similar to that described above. That is, particles were mixedwith an insulin solution to give particles containing 11.4% insulin byweight, and then the pH adjusted to promote insulin adsorption onto theparticles. The resulting particle suspensions were dried by either spraydrying or lyophilization. Table 4 shows the comparison of two 200 g lotsprepared using a commercial scale spray dryer with similar lyophilizedsamples. The bulk powders were tested for aerodynamic performance.Additional samples of bulk powders were stored at 40° C./75% RH for 15days prior to evaluation for insulin loss and formation of A21-desamidoinsulin. The spray dried powder displayed an average respirable fractionon fill (% RF/fill) of 62%; compared to an average value of 54% for thelyophilized powder. The spray-dried powder also demonstrated superiorstability. Insulin loss and A-21 formation of the spray-dried powderwere about half that of the lyophilized powder.

TABLE 4 Aerodynamics and stability of spray dried FDKP-insulin powderAndersen cascade impactor % Accelerated stability ManufacturingCartridge % RF/ % Insulin % A-21 Process % RF Emptying fill Lost FormedLyophilized 55 98 54 16.98 6.32 (average of two lots*) Spray dried 66 9462 8.83 2.63 (average of two lots*) *Lots were prepared in a similarmanner.

Example 8 Characterization of Spray-dried vs Lyophilized FDKP-insulinPowders

In a further refinement of the process, the feed temperature of the FDKPsolution was controlled. Stock solutions of fumaryl diketopiperazine(FDKP) were prepared and cooled to 11° C., 13° C., 15° C., 17° C., or19° C. and the FDKP particles were precipitated. Two differentstrategies were employed for loading and drying particles. In onestrategy, the precipitated diketopiperazine particles were washed, aninsulin solution was added and the pH adjusted to promote adsorption ofinsulin onto the particle, the mixture was frozen by dropwise additionto liquid nitrogen, and the resulting pellets were lyophilized(freeze-dried) to obtain a diketopiperazine-insulin dry powder. Inanother parallel protocol the precipitated diketopiperazine particleswere washed, an insulin solution was added, the pH adjusted, and thediketopiperazine-insulin particle suspension was spray-dried to obtain adiketopiperazine-insulin dry powder.

Two sets of replicates were prepared and the dry powders werecharacterized for aerodynamic performance (% RF/fill, cartridgeemptying, mass median aerodynamic diameter [MMAD] and geometric standarddeviation [GSD]). These data are summarized in Table 5. The % RF/fillfor these samples is shown in FIG. 9 . The stability of the powders iscompared in FIGS. 10A and 10B. As noted above, the spray-dried powdersshowed less insulin loss and less formation of A21-desamido insulin thanthe lyophilized samples.

The bulk density and tapped density of the spray-dried versus thelyophilized FDKP-insulin powder were assessed. The two sets ofreplicates were characterized for bulk and tapped density. Table 5 showsthat the spray-dried powder is more dense (by about a factor of 2) thanthe lyophilized powder. The bulk and tapped density for the spray-driedmaterials averaged 0.2 g/cc and 0.29 g/cc respectively. The bulk andtapped densities for lyophilized FDKP-insulin averaged 0.09 g/cc and0.13 g/cc respectively. These results were unexpected and surprising.This increase in density allows more powder to be placed in a singlecartridge, thereby providing for higher dosages.

TABLE 5 Effect of solution temperature on spray-dried and lyophilizedFDKP-insulin particles Solution % Bulk Tapped temperature Drying %Cartridge MMAD Density Density (° C.) method RF/fill Emptying (μm) GSD(g/cc) (g/cc) 11 spray-dried 46.0 87.0 2.8 1.9 0.171 0.260 43.8 92.1 3.21.8 0.182 0.267 lyophilized 48.4 98.4 2.7 2.1 0.077 0.121 50.2 96.1 2.42.1 0.080 0.122 13 spray-dried 57.4 93.6 2.4 2.0 0.157 0.261 54.9 93.22.2 2.0 0.156 0.260 lyophilized 55.4 94.7 2.6 1.9 0.089 0.159 54.8 95.32.3 2.1 0.077 0.143 15 spray-dried 60.2 93.9 2.2 2.1 0.153 0.254 58.394.2 2.3 2.0 0.181 0.274 lyophilized NM NM NM NM 0.078 0.127 59.8 94.52.3 2.1 0.087 0.141 17 spray-dried 62.4 94.0 2.4 2.0 0.177 0.269 61.593.4 2.2 2.0 0.186 0.291 lyophilized 52.3 96.1 2.4 2.1 0.087 0.141 58.691.8 2.2 2.1 0.083 0.134 19 spray-dried 51.6 81.8 2.4 1.8 0.179 0.27953.8 86.8 2.2 2.0 0.198 0.300 lyophilized 52.7 97.2 2.3 2.0 0.083 0.12660.4 94.7 2.4 2.1 0.100 0.142 NM—not measured

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosedherein, without departing from the scope and spirit of the invention.

Following long-standing patent law, and as used herein, the use of theword “a” or “an” when used in conjunction with the term “comprising” inthe claims and/or the specification may mean “one,” but it is alsoconsistent with the meaning of “one or more,” “at least one,” and “oneor more than one” unless specifically noted.

Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Other objects, features and advantages of the present invention willbecome apparent from the detailed description provided herein. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

What is claimed is:
 1. A process for manufacturing a crystallinemicroparticle of a diketopiperazine loaded with an active agent whereinthe diketopiperazine has the formula of2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is a succinyl,glutaryl, maleyl, or fumaryl group comprising the steps of: adding thediketopiperazine in a to water to form an aqueous suspension,maintaining the diketopiperazone solution at a controlled temperaturebetween about 15° C. to about 18° C., adding the active agent to waterto form an aqueous solution or suspension of the active, mixing thediketopiperazine aqueous suspension with the active agent aqueoussolution or suspension, and then spray-drying the admixture to removesolvent, using a spray dryer having an inlet and outlet, to form aspray-dried powder.
 2. The process of claim 1, wherein the inlet of thespray dryer has a temperature of between, or equal, to 105° C. to 140°C.
 4. The process of claim 1, wherein the spray dryer produces anatomization pressure of between, or equal, to 0.4 to 1.1 bar.
 5. Theprocess of claim 1, wherein the spray dryer produces a spray rate ofbetween, or equal to, 4.4 to 12.2 g/min.
 6. The process of claim 1,wherein the spray dryer produces a spray-dried powder of between, orequal to, 0.150 to 0.200 g/cc.
 7. The process of claim 1, wherein thespray dryer produces a spray-dried powder that has a tap density ofbetween, or equal to, 0.250 to 0.300 g/cc.
 8. The process of claim 1,wherein the active agent is insulin.
 9. The process of claim 8, whereinthe spray-dried powder has an insulin content of about 3% to about 50%by weight.
 10. The process of claim 1, wherein the active agent isselected from the group consisting of: insulin, calcitonin, parathyroidhormone 1-34, or other bioactive fragment of parathyroid hormone,octreotide, leuprolide, and RSV peptide, felbamate, cannabinoidantagonists and/or agonists, muscarinic antagonists and/or agonists,heparin, low molecular weight heparin, cromolyn, sildenafil, vardenafil,tadalafil growth hormone, zidovudine (AZT), didanosine (DDI),granulocyte-colony stimulating factor (GCSF), lamotrigine, chorionicgonadotropin releasing factor, luteinizing release hormone,β-galactosidase, GLP-1, exendins 1-4, ghrelin, and fragments thereof.